Does intersex matter? A case study of rainbow darter in the Grand River

Endocrine disrupting compounds (EDCs) are present in the environment and can have negative effects on the health of wildlife. Aquatic organisms residing near the outfalls of municipal wastewater effluent (MWWE) are chronically exposed to EDCs, including natural hormones, pharmaceuticals, and industrial chemicals. The vulnerability of aquatic organisms to these compounds is due to the evolutionary conservation of endocrine systems. Although numerous studies have indicated that compounds in MWWE including estrogenic and anti-androgenic contaminants, feminize male fish, it is still uncertain what the consequences of feminization of male fish are. Research on this topic since the early 1990’s has demonstrated that a multitude of compounds in MWWE are capable of binding to estrogen receptors in fish. Key biomarkers of estrogen exposure are the elevation of vitellogenin protein and gene expression levels, as well as the presence of female tissue in male gonads; a condition referred to as intersex. The feminization of male fish and intersex condition has been noted in populations of fish around the world including rainbow darter (Etheostoma caeruleum) in the Grand River, Ontario, Canada. Male rainbow darter collected from sites near MWWE outfalls have intersex condition as well as impaired androgen production and sperm development. The aim of the studies in this thesis was to assess the implications of feminized males using rainbow darter as a model. Several questions were addressed through field and laboratory studies. The first question addressed was whether exposure to MWWE, and intersex in particular, reduced the reproductive success of rainbow darter. To test this, fish were collected in the field during the spawning season, gametes were stripped, and manual fertilizations conducted at each of several sites. Fertilization success and embryo survival were lower at sites downstream of a MWWTP outfall when compared to a rural reference site. Additionally, when grouped into categories based on the severity of intersex (based on number and development of oocytes in testes), it was found that the most severely affected males had the lowest fertilization success. In contrast, no relationship was found between embryo survival and intersex severity, suggesting that egg quality may play a larger role in the survival of embryos. This study concluded that severe intersex condition, which is a marker of MWWE exposure, was an indicator of poor reproductive success in male rainbow darter. The second question addressed in this thesis was whether reproductive behaviour was altered in fish exposed to MWWE. To test this, rainbow darter were collected from sites in the Grand River along an urban gradient and two separate experiments conducted. The first experiment was a breeding competition experiment where three males, each from a different site, were placed into a spawning tank with a single female from either a reference or MWWE exposed site. Reproductive behaviour of both male and female rainbow darter collected near MWWTP outfalls were impaired. Males spent less time guarding the spawning area and females performed fewer nose digs. Both behaviours are important for reproductive success. The second experiment in this study assessed the response of males to a mirror-competitor. Males with severe intersex condition spent less time performing aggressive acts than males with no intersex condition. From these findings, this study concluded that MWWE exposure, as indicated by intersex severity in the second experiment, alters reproductive behaviour of rainbow darter and would likely lead to reduced reproductive fitness in the wild. The third study in this thesis tested whether observations of impaired reproductive success and behaviour are directly associated with MWWE as opposed to other urban stressors. In this study, male and female rainbow darter were collected from a reference site during the pre-spawning period and placed in breeding groups. Following a 10-day pre-exposure spawning period, rainbow darter breeding groups were exposed to 1 of 5 treatments (control, 17α-ethinylestradiol (EE2, a synthetic estrogen), 1%, 10%, or 20% MWWE) for 21 days. After the 21-day exposure, male behaviour was assessed in a mirror-competitor test. Fecundity (number of eggs) was lower in the highest (20%) dose of MWWE during the first week of the exposure but was comparable to other groups for the remainder of the experiment. Fertilization success was also lower during the exposure period compared to the pre-exposure period in MWWE exposed and EE2 exposed fish. Males exposed to 20% MWWE were less aggressive and more active than the control group. Although the responses of this experiment were less dynamic than those observed in fish exposed in the wild, they corroborated the findings that rainbow darter exposed to MWWE have reduced reproductive fitness. The final study in this thesis addressed the issue of repeatability of field studies of feminized males by comparing the variability of biological measures between fall and spring seasons among five years of collections as well as across levels of biological organization. Measures were most consistent in males collected during the fall. Additionally, measures at the tissue level, including sperm development and intersex incidence and severity, were the most consistent measures among years and seasons. This study concluded that the use of multiple biological measures from several levels of biological organization allowed for the same conclusion to be made about the reproductive health of rainbow darter in all years and both seasons of field collections. Throughout these studies, intersex was found to be one of the most reliable markers of exposure to endocrine disrupting compounds in municipal wastewater effluent. Additionally, from the sum of these studies, it can be concluded that highly prevalent and severe intersex in a population of fish indicates poor reproductive fitness. Whether this results in reduced population size or decreased genetic diversity is not known and is an important topic for future studies

Ferredoxin 1b (Fdx1b) Is the essential mitochondrial redox partner for cortisol biosynthesis in zebrafish

Mitochondrial cytochrome P450 (CYP) enzymes rely on electron transfer from the redox partner ferredoxin 1 (FDX1) for catalytic activity. Key steps in steroidogenesis require mitochondrial CYP enzymes and FDX1. Over 30 ferredoxin mutations have been explored in vitro; however, no spontaneously occurring mutations have been identified in humans leaving the impact of FDX1 on steroidogenesis in the whole organism largely unknown. Zebrafish are an important model to study human steroidogenesis, because they have similar steroid products and endocrine tissues. This study aimed to characterize the influence of ferredoxin on steroidogenic capacity in vivo by using zebrafish. Zebrafish have duplicate ferredoxin paralogs: fdx1 and fdx1b. Although fdx1 was observed throughout development and in most tissues, fdx1b was expressed after development of the zebrafish interrenal gland (counterpart to the mammalian adrenal gland). Additionally, fdx1b was restricted to adult steroidogenic tissues, such as the interrenal, gonads, and brain, suggesting that fdx1b was interacting with steroidogenic CYP enzymes. By using transcription activator-like effector nucleases, we generated fdx1b mutant zebrafish lines. Larvae with genetic disruption of fdx1b were morphologically inconspicuous. However, steroid hormone analysis by liquid chromatography tandem mass spectrometry revealed fdx1b mutants failed to synthesize glucocorticoids. Additionally, these mutants had an up-regulation of the hypothalamus-pituitary-interrenal axis and showed altered dark-light adaptation, suggesting impaired cortisol signaling. Antisense morpholino knockdown confirmed Fdx1b is required for de novo cortisol biosynthesis. In summary, by using zebrafish, we generated a ferredoxin knockout model system, which demonstrates for the first time the impact of mitochondrial redox regulation on glucocorticoid biosynthesis in vivo

Sustained Action of Developmental Ethanol Exposure on the Cortisol Response to Stress in Zebrafish Larvae and Adults

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedThis study was supported by the National Centre for the replacement, refinement and reduction of animals in research

Reduction of intersex in a wild fish population in response to major municipal wastewater treatment plant upgrades

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Environmental Science & Technology, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see https://doi.org/10.1021/acs.est.6b05370Intersex in fish downstream of municipal wastewater treatment plants (MWWTPs) is a global concern. Consistent high rates of intersex in male rainbow darter (Etheostoma caeruleum) have been reported for several years in the Grand River, in southern Ontario, Canada, in close proximity to two MWWTPs. The larger MWWTP (Kitchener) recently underwent upgrades that included the conversion from a carbonaceous activated sludge to nitrifying activated sludge treatment process. This created a unique opportunity to assess whether upgrades designed to improve effluent quality could also remediate the intersex previously observed in wild fish. Multiple years (2007–2012) of intersex data on male rainbow darter collected before the upgrades at sites associated with the MWWTP outfall were compared with intersex data collected in postupgrade years (2013–2015). These upgrades resulted in a reduction from 70 to 100% intersex incidence (preupgrade) to <10% in postupgrade years. Although the cause of intersex remains unknown, indicators of effluent quality including nutrients, pharmaceuticals, and estrogenicity improved in the effluent after the upgrades. This study demonstrated that investment in MWWTP upgrades improved effluent quality and was associated with an immediate change in biological responses in the receiving environment. This is an important finding considering the tremendous cost of wastewater infrastructure

Biological responses in fish exposed to municipal wastewater treatment plant effluent in situ

Effluents from municipal wastewater treatment plants (MWTPs) are complex mixtures of chemicals including endocrine-disrupting compounds (EDCs) and 17α-ethynylestradiol (EE2). The objective of this study was to evaluate selected responses of two fish species, in two different years, exposed in situ to MWTP effluent. Biological markers of exposure (plasma vitellogenin (VTG) and antioxidant enzymes) were measured in two species of male fish, rainbow trout (Oncorhynchus mykiss) and fathead minnow (Pimephales promelas), caged at sites associated with wastewater outfall. The estrogenicity of the final effluent in 2010 was determined to be 17.0 + 0.4 ng/L estrogen equivalents (EEQ) and reduced to 7.5 + 2.9 ng/L EEQ after infrastructure upgrades. Pharmaceuticals and personal care products in the effluent and surface water in both years confirmed the exposures at each downstream site. Despite the presence of estrogenic compounds in the MWTP effluent, no effluent-caged male fish demonstrated plasma VTG induction. Minnows and trout that received an intraperitoneal injection of 5 mg/g EE2 showed VTG induction at both field sites. In 2012, the liver somatic index (LSI) of both species increased with exposure, as did changes in antioxidant enzymes, and reactive oxygen species (ROS) activity. Multiple biological mechanisms are modified by effluent exposure, and multiple endpoints are needed to assess risk.Standards Development Branch|| Ontario Ministry of the Environment and Climate Change (OMECC)||the Canadian Water Network||Natural Sciences and Engineering Research Council|| Canada Research Chairsto MRS|| OMECC Laboratory Services Branch||Trent University||University of Ottawa ||Environment and Climate Change Canad

Tracking emergence and spread of SARS-CoV-2 omicron variant in large and small communities by wastewater monitoring in Alberta, Canada

Wastewater monitoring of SARS-CoV-2 enables early detection and monitoring of the COVID-19 disease burden in communities and can track specific variants of concern. We determined proportions of the Omicron and Delta variants across 30 municipalities covering >75% of the province of Alberta (population 4.5 million), Canada, during November 2021-January 2022. Larger cities Calgary and Edmonton exhibited more rapid emergence of Omicron than did smaller and more remote municipalities. Notable exceptions were Banff, a small international resort town, and Fort McMurray, a medium-sized northern community that has many workers who fly in and out regularly. The integrated wastewater signal revealed that the Omicron variant represented close to 100% of SARS-CoV-2 burden by late December, before the peak in newly diagnosed clinical cases throughout Alberta in mid-January. These findings demonstrate that wastewater monitoring offers early and reliable population-level results for establishing the extent and spread of SARS-CoV-2 variants

Near real-time determination of B.1.1.7 in proportion to total SARS-CoV-2 viral load in wastewater using an allele-specific primer extension PCR strategy

"The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome corona- virus 2 (SARS-CoV-2) has claimed millions of lives to date. Antigenic drift has resulted in viral variants with putatively greater transmissibility, virulence, or both. Early and near real-time detection of these variants of concern (VOC) and the ability to accurately follow their incidence and prevalence in communities is wanting. Wastewater-based epidemiology (WBE), which uses nucleic acid amplification tests to detect viral fragments, is a reliable proxy of COVID-19 incidence and prevalence, and thus offers the potential to monitor VOC viral load in a given population. Here, we describe and validate a primer extension PCR strategy targeting a signature mutation in the N gene of SARS-CoV-2. This allows quantification of B.1.1.7 versus non-B.1.1.7 allele frequency in wastewater without the need to employ quantitative RT-PCR standard curves. We show that the wastewater B.1.1.7 profile correlates with its clinical counterpart and benefits from a near real-time and facile data collection and reporting pipeline. This assay can be quickly implemented within a current SARS-CoV-2 WBE framework with minimal cost; allowing early and contemporaneous estimates of B.1.1.7 community transmission prior to, or in lieu of, clinical screening and identification. Our study demonstrates that this strategy can provide public health units with an additional and much needed tool to rapidly triangulate VOC incidence/prevalence with high sensitivity and lineage specificity"National Microbiology Laboratory||Water Services at the Cities of Ottawa and Barrie||Ottawa Public Health||Simcoe Muskoka District Health Unit|| Public Health Ontario||Ontario Wastewater Surveillance Initiativ

Multiplex RT-qPCR assay (N200) to detect and estimate prevalence of multiple SARS-CoV-2 Variants of Concern in wastewater

Wastewater-based surveillance (WBS) has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Quantities of viral fragments of SARS-CoV-2 in wastewater are related to numbers of clinical cases of COVID-19 reported within the corresponding sewershed. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription quantitative polymerase chain reaction (RT-qPCR) or sequencing. A multiplex RT-qPCR assay to detect and estimate the prevalence of multiple VOCs, including Omicron/Alpha, Beta, Gamma, and Delta, in wastewater RNA extracts was developed and validated. The probe-based multiplex assay, named “N200” focuses on amino acids 199-202, a region of the N gene that contains several mutations that are associated with variants of SARS- CoV-2 within a single amplicon. Each of the probes in the N200 assay are specific to the targeted mutations and worked equally well in single- and multi-plex modes. To estimate prevalence of each VOC, the abundance of the targeted mutation was compared with a non- mutated region within the same amplified region. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from six sewersheds in Ontario, Canada collected between December 1, 2021, and January 4, 2022. Using the N200 assay, the replacement of the Delta variant along with the introduction and rapid dominance of the Omicron variant were monitored in near real-time, as they occurred nearly simultaneously at all six locations. The N200 assay is robust and efficient for wastewater surveillance can be adopted into VOC monitoring programs or replace more laborious assays currently being used to monitor SARS- CoV-2 and its VOCs.Ontario Ministry of the Environment, Conservation and Parks||Natural Sciences and Engineering Research Council of Canad

The Role of Wastewater Testing for SARS-CoV-2 Surveillance

Wastewater testing for SARS-CoV-2 is relatively new; however, it builds on existing public health surveillance infrastructure. There is a limited but growing evidence base for its use, despite notable interpretation challenges. Wastewater testing results have helped to inform public health policy and interventions during the COVID-19 pandemic in Ontario and other jurisdictions. Wastewater testing for SARS-CoV-2 is useful for early detection of outbreaks and surges as well as population-wide surveillance of COVID-19 that is complementary to clinical testing. Further, it offers an efficient means of SARS-CoV-2 surveillance for specific settings such as correctional facilities, shelters, and university residences. Wastewater testing can also be used for the detection and monitoring of variants of concern (VOCs)

Wastewater to clinical case (WC) ratio of COVID-19 identifies insufficient clinical testing, onset of new variants of concern and population immunity in urban communities

Clinical testing has been the cornerstone of public health monitoring and infection control efforts in communities throughout the COVID-19 pandemic. With the extant and anticipated reduction of clinical testing as the disease moves into an endemic state, SARS-CoV-2 wastewater surveillance (WWS) is likely to have greater value as an important diagnostic tool to inform public health. As the widespread adoption of WWS is relatively new at the scale employed for COVID-19, interpretation of data, including the relationship to clinical cases, has yet to be standardized. An in-depth analysis of the metrics derived from WWS is required for public health units/agencies to interpret and utilize WWS-acquired data effectively and efficiently. In this study, the SARS-CoV-2 wastewater signal to clinical cases (WC) ratio was investigated across seven different cities in Canada over periods ranging from 8 to 21 months. Significant increases in the WC ratio occurred when clinical testing eligibility was modified to appointment-only testing, identifying a period of insufficient clinical testing in these communities. The WC ratio decreased significantly during the emergence of the Alpha variant of concern (VOC) in a relatively non-immunized community’s wastewater (40-60% allelic proportion), while a more muted decrease in the WC ratio signaled the emergence of the Delta VOC in a relatively well-immunized community’s wastewater (40-60% allelic proportion). Finally, a rapid and significant decrease in the WC ratio signaled the emergence of the Omicron VOC, likely because of the variant’s greater effectiveness at evading immunity, leading to a significant number of new reported clinical cases, even when vaccine-induced community immunity was high. The WC ratio, used as an additional monitoring metric, complements clinical case counts and wastewater signals as individual metrics in its ability to identify important epidemiological occurrences, adding value to WWS as a diagnostic technology during the COVID-19 pandemic and likely for future pandemics.Ontario's Ministry of Environment, Conservation and Parks||Alberta Healt